Fixed negative charges in the glomerular basement membrane (GBM) are important in restricting anionic plasma proteins from passing into the urine and these charges are lost during nephrosis as proteinuria develops. These sites, when stained with alcian blue, appear as 90 A fibrils in the laminae rarae of the GBM. The chemical natures of these sites is undetermined. This proposal seeks to develop a murine tumor, which has been shown to contain collagen similar to that found in basement membranes as a model of the rat GBM. In particular, it will be examined for anionic sites similar to those seen in GBM. These sites will be extracted and characterized by polyacrylamide gel electrophoresis and histochemical techniques. These experiments will determine what the anionic sites are. In addition, whole tumor extracts as well as pepsin and collagenase digests of these extracts will be used to immunize mice, rats, rabbits and lambs in an attempt to produce anti-GBM nephritis. These animals will be examined by electron and immunofluorescence microscopy. In addition, they will be tested for proteinuria and antibodies to tumor proteins. These antibodies will be purified by immune adsorption. An additional attempt will be made to produce pure antibodies using hybridoma techniques. These purified antibodies will be tested for nephrotoxicity by injecting them into heterologous and homologous species. The animals will be examined microscopically to see whether the collagenous or non-collagenous antibodies are producing the greater lesions in the heterologous phase. With the hybridoma antibodies this study may be done with different IgG subclasses. In addition, these antibodies will be used to determine the distribution of the various components of the GBM using ferritin or horseradish peroxidase conjugated antibodies in direct or indirect immunocytochemical techniques. Finally, arthritic synovium from rats immunized with type II collagen will be examined by both transmission and scanning microscopy in order to establish the sequence of pathological changes in the disease. This model is relevant to human disease because human patients have cellular sensitivitiy to type II collagen.